Tubulysin analogues

ABSTRACT

The invention relates to tubulysin derivatives of general formula (II), said derivatives having a cytostatic effect.

The present invention refers to novel tubulysin analogs and its use forthe treatment of cancer diseases.

Tubulysins, for the first time were isolated by Höfle and Reichenbach etal. (GBF Braunschweig) from a culture growth of the myxobacterialstrains of Archangium gephyra (F. Sasse et al. J. Antibiot. 2000, 53,879-885; WO9813375; DE 10008089). These compounds show high cytotoxicityin the low picomolare IC₅₀ in a panel of cancer cell lines; thus theyare of interest as potential anticancer therapeutics. Tubulysins (I) aretetrapeptides, containing three unusual amino acids; thus the totalsynthesis pose a considerable challenge to organic chemists.

-   Tubulysin A: R′=CH₂CH(CH₃)₂; R″=OH-   Tubulysin B: R′=CH₂CH₂CH₃; R″=OH-   Tubulysin C: R′=CH₂CH₃; R″=OH-   Tubulysin D: R′=CH₂CH(CH₃)₂; R″=H-   Tubulysin E: R′=CH₂CH₂CH₃; R″=H-   Tubulysin F: R′=CH₂CH₃; R″=H

It is an objective of the present invention to provide novel Tubulysinanalogues with improved activity and properties, in particularpharmacological properties as compared to the natural products.

The present invention provides a compound of Formula (II):

wherein

-   A is a substituted 5- or 6-membered heteroaryl;-   wherein-   X is O, S or NR¹³ or CR¹⁴R¹⁵;-   wherein-   Y is O, S or NR¹⁶;-   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,    R¹⁶ are independently H, alkyl, alkenyl, alkinyl, heteroalkyl, aryl,    heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl,    heterocycloalkyl, aralkyl or heteroaralkyl, or two R's are members    of a cycloalkyl or heterocycloalkyl ring system;-   wherein compounds of Formula (I) are excluded,-   wherein R′ are H, alkyl, alkenyl, aryl or heteroaryl and—at the same    time—R″ are H, —OH, alkyl, aryl, or heteroaryl;    or a pharmacologically acceptable salt, a solvate, a hydrate or a    pharmacologically acceptable formulation thereof. Explicitly    excluded are Tubulysins A, B, C, D, E and F.

The term alkyl or alk refers to a saturated, linear or branchedhydrocarbon group, containing from one to twenty carbon atoms,preferably from one to twelve carbon atoms, mostly preferred from one tosix carbon atoms, for example methyl, ethyl, propyl, isopropyl,isobutyl, n-butyl, tert-butyl, n-hexyl, 2,2-dimethylbutyl or n-octyl.

The term alkenyl and alkinyl refers to a at least partially unsaturated,linear or branched hydrocarbon group, containing from two to twentycarbon atoms, preferably from two to twelve carbon atoms, mostlypreferred from two to six carbon atoms, for example ethenyl, allyl,acetylenyl, prpargyl, isoprenyl, or hex-2-enyl. Preferentially alkenylgroups contain one or two, mostly preferred one double bond and alkinylgroup contain one or two, mostly preferred one triple bond.

Optionally the term akyl, alkenyl and alkinyl refers to groups where oneor several, preferentially one, two or three hydrogen atoms are replacedby a halogen atom, preferentially fluorine or chlorine or a2,2,2-trichlorethyl, or a trifluoromethyl.

The term heteroalkyl refers to a alkyl, alkenyl or alkinyl group, whereseveral, preferentially one, two or three carbon atoms are replaced by aO, N, P, B, Se, Si, or S atom, preferentially O, S, N. The termheteroalkyl refers to a carboxylic acid or a thereof derived group, forexample acyl (alkyl-CO), acylalkyl, alkoxycarbonyl, acyloxy,acyloxyalkyl, carboxyalkylamid or alkoxycarbonyloxy.

Examples of heteroalkyl groups are groups of the formula R^(a)—O—Y^(a)—,R^(a)—S—Y^(a)—, R^(a)—N(R^(b))—Y^(a)—, R^(a)—CO—Y^(a)—,R^(a)—O—CO—Y^(a)—, R^(a)—CO—O—Y^(a)—, R^(a)—CO—N(R^(b))—Y^(a)—,R^(a)—N(R^(b))—CO—Y^(a)—, R^(a)—O—CO—N(R^(b))—Y^(a)—,R^(a)—N(R^(b))—CO—O—Y^(a)—, R^(a)—N(R^(b))—CO—N(R^(c))—Y^(a)—,R^(a)—O—CO—O—Y^(a)—, R^(a)—N(R^(b))—C(═NR^(d))—N(R^(c))—Y^(a)—,R^(a)—CS—Y^(a)—, R^(a)—O—CS—Y^(a)—, R^(a)—CS—O—Y^(a)—,R^(a)—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—Y^(a)—,R^(a)—O—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—O—Y^(a)—,R^(a)—N(R^(b))—CS—N(R^(c))—Y^(a)—, R^(a)—O—CS—O—Y^(a)—,R^(a)—S—CO—Y^(a)—, R^(a)—CO—S—Y^(a)—, R^(a)—S—CO—N(R^(b))—Y^(a)—,R^(a)—N(R^(b))—CO—S—Y^(a)—, R^(a)—S—CO—O—Y^(a)—, R^(a)—O—CO—S—Y^(a)—,R^(a)—S—CO—S—Y^(a)—, R^(a)—S—CS—Y^(a)—, R^(a)—CS—S—Y^(a)—,R^(a)—S—CS—N(R^(b))—Y^(a)—, R^(a)—N(R^(b))—CS—S—Y^(a)—,R^(a)—S—CS—O—Y^(a)—, R^(a)—O—CS—S—Y^(a)—, wherein R^(a) refers to a H, aC₁-C₆-alkyl, a C₂-C₆-alkenyl or a C₂-C₆-alkinyl group; wherein R^(b)refers to a H, a C₁-C₆-alkyl, a C₂-C₆-alkenyl or a C₂-C₆-alkinyl group;wherein R^(c) refers to a H, a C₁-C₆-alkyl, a C₂-C₆-alkenyl or aC₂-C₆-alkinyl group; wherein R^(d) refers to a H, a C₁-C₆-alkyl, aC₂-C₆-alkenyl or a C₂-C₆-alkinyl group and Y^(a) refers to a directbinding, a C₁-C₆-alkylen, a C₂-C₆-alkenylen or a C₂-C₆-alkinylen group,wherein each heteroalkyl group can be replace by a carbon atom and oneor several hydrogen atoms can be replaced by fluorine or chlorine atoms.Examples of heteroalkyl groups are methoxy, trifluormethoxy, ethoxy,n-propyloxy, iso-propyloxy, tert-butyloxy, methoxymethyl, ethoxymethyl,methoxyethyl, methylamino, ethylamino, dimethylamino, diethylamino,iso-propylethylamino, methyl-aminomethyl, ethylaminomethyl,di-iso-propylaminoethyl, enolether, dimethylaminomethyl,dimethylaminoethyl, acetyl, propionyl, butyryloxy, acetyloxy,methoxycarbonyl, ethoxy-carbonyl, N-ethyl-N-methylcarbamoyl orN-methylcarbamoyl. Other examples of heteroalkyl groups are nitrile,isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate andalkylnitrile groups.

The term cycloalkyl refers to a saturated or partially unsaturated (e.g.cycloalkenyl) cyclic group, comprising one or several rings,preferentially one or two, containing three to fourteen ring carbonatoms, preferentially three to ten, preferentially three, four, five,six or seven ring carbon atoms. Furthermore the term cycloalkyl refersto a group where one or more hydrogen atoms are replaced by F, Cl, Br,I, OH, ═O, SH, ═S, NH₂, ═NH, or NO₂, or cyclic ketones, for examplecyclohexanone, 2-cyclohexenone or cyclopentanone. Examples of cycloalkylgroups are cyclopropyl, cyclobutyl, cyclopentenyl, spiro[4,5]-decanyl,norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl,cubanyl, bicyclo[4.3.0]nonyl, tetralin, cyclopentylcyclohexyl,fluor-cyclohexyl or the cyclohex-2-enyl group.

The term heterocycloalkyl refers to the above definition, wherein a orseveral, preferentially one, two or three ring carbon atoms are replacedby a O, N, Si, Se, P, or S, preferentially O, S, N. Preferentially aheterocycloalkyl groups is composed of one or two rings comprising threeto ten, preferentially three, four, five, six or seven ring atoms.Moreover the term heterocycloalkyl refers to groups where a or severalhydrogen atoms are replaced by F, Cl, Br, I, OH, ═O, SH, ═S, NH₂, NO₂.Examples of heterocycloalkyl are piperidyl, morpholinyl, urotropinyl,pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydro-furyl,oxacyclopropyl, azacyclopropyl or 2-pyrazolinyl groups as well aslactams, lactons, cyclic imides and cyclic anhydrides.

The term alkylcycloalkyl refers to groups, which contain cycloalkyl aswell as alkyl, alkenyl or alkinyl groups according to the abovedefinition, e.g. alkylcycloalkyl, alkylcycloalkenyl, alkenylcycloalkyland alkinylcycloalkyl groups. Preferentially a alkylcycloalkyl group iscomposed of a cycloalkyl group, comprising one or more rings, comprisingthree to ten, preferentially three, four, five, six or sevencarbon—atoms and one or two alkyl, alkenyl other alkinyl groups with oneor two to six carbon atoms.

The term heteroalkylcycloalkyl refers to alkylcycloalkyl groups,according to the above definition, wherein one or several,preferentially one, two or three carbon atoms are replaced by O, N, Si,Se, P or S, preferentially O, S, N. Preferentially it is composed of aheteroakylcycloalkyl group comprising one or two ring systems with threeto ten, preferentially three, four, five, six or seven ring atoms andone or two alkyl, alkenyl, alkinyl or heteroalkyl groups with one or twoto six carbon atoms. Examples of such a group are alkylheterocycloalkyl,alkylheterocycloalkenyl, alkenyl-heterocycloalkyl,alkinylheterocycloalkyl, heteroalkyl-cycloalkyl,heteroalkylheterocycloalkyl and heteroalkylheterocylcloalkenyl, whereinthe cyclic group is saturated or partially (simply, twofold orthreefold) unsaturated.

The term aryl or ar refers to a aromatic group, composed of one orseveral rings, comprising six to fourteen carbon atoms, preferentiallysix to ten, preferentially six carbon atoms. The term aryl or ar refersto a aromatic group, wherein one or several H atoms are replaced by F,Cl, Br or I or OH, SH, NH₂, or NO₂. Examples are phenyl-, naphthyl-,piphenyl-, 2-fluorphenyl, anilinyl-, 3-nitrophenyl or 4-hydroxy-phenyl.

The term heteroaryl refers to a aromatic group, composed of one orseveral rings, comprising five to fourteen rind atoms, preferentiallyfive to ten, and a or several, preferentially one, two, three or four O,N, P or S ring atoms, preferentially O, S or N. The term heteroarylrefers to groups, wherein one or several H atoms are replaced by F, Cl,Br or I or OH, SH, NH₂, or NO₂. Examples are 4-pyridyl, 2-imidazolyl,3-phenylpyrrolyl, thiazolyl, oxazolyl, triazolyl, tetrazolyl,isoxazolyl, indazolyl, indolyl, benzimidazolyl, pyridazinyl, chinolinyl,purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3′-bifuryl, 3-pyrazolyl andiso-chinolinyl.

The term aralkyl refers to groups, in accordance to the abovedefinition, composed of aryl and alkyl, alkenyl, alkinyl and/orcycloalkyl, e.g. arylalkyl, arylalkenyl, arylalkinyl, arylcycloalkyl,arylcycloalkenyl, alkylarylacycloalkyl and alkylarylcycloalkenyl.Examples of aralkyles are toluol, xylol, mesitylen, styren,benzylchloride, o-fluortoluene, 1H-inden, tetralin, dihydronaphthaline,indanon, phenylcyclopentyl, cumol, cyclo-hexylphenyl, fluoren and indan.Preferentially, a aralkyl group is composed of composed of one or twoaromatic rings, comprising six to ten ring carbon atoms and one or twoalkyl, alkenyl and/or alkinyl comprising one or two to six carbon atomsand/or one cyclo-alkyl comprising five or six ring carbon atoms.

The term heteroaralkyl refers to groups, in accordance to the abovedefinition, wherein one or several, preferentially one, two, three orfour carbon atoms are replaced by O, N, Si, Se, P, B, S, preferentiallyO, N or S, and groups which according to the above definition containaryl, heteroaryl and alkyl, alkenyl, alkinyl and/or heteroalkyl and/orcycloalkyl and/or heterocyclo-alkyl. Preferentially a heteroaralkylgroup is composed od a or two aromatic ring systems comprising five orsix to ten carbon atoms and one or two alkyl, alkenyl and/or alkinylcomprising one or two to six carbon atoms and/or one cycloalkylcomprising five or six ring carbon atoms, wherein one, two, three orfour carbon atoms can be replaced by O, N or S.

Examples are arylheteroalkyl, arylheterocycloalkyl,arylheterocycloalkenyl, arylalkylheterocycloalkyl,arylalkenylheterocycloalkyl, arylalkinylheterocyclo-alkyl,arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkinyl, heteroarylheteroalkyl, heteroarylcycloalkyl,heteroarylcycloalkenyl, heteroarylheterocycloalkyl,heteroarylheterocycloalken-yl, heteroarylalkylcycloalkyl,heteroarylalkylhetero-cycloalkenyl, heteroarylheteroalkylcycloalkyl,Hetero-arylheteroalkylcycloalkenyl and heteroarylheteroalkylheterocycloalkyl, wherein the cyclic groups can be saturated or simple,twice, three fold of four fold unsaturated. Examples aretetrahydroisochinolinyl, benzoyl, 2- or 3-ethyl-indolyl,4-methylpyridino, 2-, 3- or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or4-carboxyphenylalkyl.

The terms cycloalkyl, heterocycloalkyl, alkylcyclo-alkyl,heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl referto groups, wherein one or several H atoms are replaced by F, Cl, Br or Ior OH, SH, NH₂, or NO₂.

The term “optinally substituiert” relates to groups, wherein one orseveral H atoms are replaced by F, Cl, Br or I or OH, SH, NH₂, or NO₂.The term “gegebenenfalls substituiert” relates further to groups,comprising exclusively or in addition unsubstituted C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkinyl, C₁-C₆ heteroalkyl, C₃-C₁₀ cycloalkyl, C₂-C₉heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₉ heteroaryl, C₇-C₁₂ aralkyl orC₂-C₁₁ heteroaralkyl.

Protecting groups are known to the specialist and described in P. J.Kocienski, Protecting Groups, Georg Thieme Verlag, Stuttgart, 1994 andin T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis,John Wiley & Sons, New York, 1999. Common amino protecting groups aree.g. t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz, Z), benzyl (Bn),benzoyl (Bz), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl(Alloc), trichlorethyloxycarbonyl (Troc), acetyl or trifluoracetyl.

Compounds of Formula (II) can comprise several chiral centers related totheir substitution pattern. The present invention relates to all aldefined enantio and diastereo isomers as well as their mixtures in allratios. Moreover the present invention relates to all cis/trans isomersof compounds of the general Formula (II) as well as their mixtures.Moreover the present invention relates to all tautomeric forms ofcompounds of the general Formula (II).

Preferably A constitutes a optinally substituted thizol ring; morepreferably A has the following structure:

Moreover preferably X constitutes a CH₂ group.

Preferably Y constitutes O.

Preferably R¹ constitutes a C₁-C₄ alkyl.

Preferably R² ans R³ constitute together (CH₂)_(n) with n=2, 3, 4 or 5.

Preferably R⁴ constitutes H or methyl.

Preferably R⁵ constitutes H.

Preferably R⁵ constitutes C₁-C₆ alkyl, C₃-C₆ cycloalkyl or C₄-C₇lkylcycloalkyl.

Preferably R⁵ constitutes H or methyl.

Preferably R⁸ constitutes CH₂OCOR¹⁷, wherein R¹⁷ constitutes C₁-C₆ alkylor C₁-C₆ alkenyl.

Preferably R⁹ constitutes C₁-C₆ alkyl.

Preferably R¹⁰ constitutes H or methyl.

Preferably R¹¹ constitutes H or —(C═O)—(C₁₋₄)Alkyl.

Preferably R¹² constitutes NR¹⁸R¹⁹, wherein R¹⁸ constitutes H or methyland R¹⁹ constitutes aralkyl or heteroaralkyl.

Most preferably are compounds of Formula (III),

wherein R¹ comprise C₁-C₄ alkyl, R⁶ comprise C₁-C₆ alkyl, R⁹ compriseC₁-C₆ alkyl, R¹⁷ comprise C₁-C₆ alkyl or C₁-C₆ alkenyl, R¹⁹ comprisearalkyl or heteroaralkyl, R²⁰ comprise C₁-C₄ alkyl and m equals 1 or 2.

Preferentially R¹⁹ comprise the following structure:

wherein R²¹ comprise OH, NH₂, alkyloxy, alkyl amino or dialkyl amino,R²² comprise halogen, OH, NO₂, NH₁, alkyloxy, alkyl amino or dialkylamino and p equals 0, 1, 2 or 3.

Examples of pharmacologically acceptable salts of compounds of Formula(II) are physiologically acceptable mineral acids, e.g. hydrochloricacid, sulfuric acid, phorphoric acid or salts of organic acids, e.g.methansulfonic acid, p-toluenesulfonic acid, lactic acid, formic acid,trifluoracetic acid, citric acid, succinic acid, fumaric acid, maleicacid and salicylic acid. Compounds of Formula (II) can be solvated,especially hydrated. The hydration can occur during the synthesisprocess or can be a consequence of the hygroscopic nature of theoriginally dehydrated compound of Formula (II). Compounds of Formula(II), containing assymetric carbon atoms might exist as mixtures ofdiastereomers, as mixtures of enantiomers or as optically purecompounds.

The pharmaceutical composition according to the present invention iscomposed of at least one compound of Formula (II) and optinally carrierand/or adjuvants.

Pro drugs are also subject of the present invention and they arecomposed of a compound of Formula (II) and at least onepharmacologically acceptable protecting group, which is cleaved underphysiological conditions, e.g. alkoxy, aralkyloxy, acyl or acyloxy, moreprecisely ethoxy, benzyloxy, acetyl or acetyloxy. Moreover the presentinvention relates to conjugates comprising at least one compound ofFormula (II) and a biological macromolecule, e.g. oligo saccharide,monoclonale antibody, lectine, PSA (prostate specific antigen) orpeptidic vectors and if needed as well as a suitable linker. Theexpression linker relates to a chemical group, which links compounds ofFormula (II) with a biological macromolecule. Examples of linkers arealkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,aralkyl or heteroaralkyl.

The therapeutic usage of compounds of Formula (II), its pharmacologicacceptable salts and/or its solvates and hydrates, as well as thecorresponding formulations and pharmacological compositions are alsosubject of the present invention.

The usage of the active agents for the preparation of drugs for thetreatment of cancer is also subject of the present invention. Moreoverthe present compounds are of interest for the prevention and/ortreatment of rheumatoid arthritis, inflammatory diseases, immunologicaldiseases (e.g. type I diabetes), autoimmune diseases, other tumordiseases as well as for the surface treatment (impregnation) of plasticand metal implants, e.g. stents. In general, compounds of Formula (II)will be given as a single treatment or in combination with an arbitrarytherapeutic substance according to known and accepted modes. Suchtherapeutically useful compositions can be administered in one of thefollowing ways: orally, including dragees, coated tablets, pills,semi-solids, soft or hard capsules, solutions, emulsions or suspensions;parenteral, including injectable solutions; rectal as suppositories; byinhalation, including powder formulation or as a spray, transdermal orintranasal. For the production of such tablets, pills, semi solids,coated tablets, dragees and hard gelatine capsules the therapeuticallyused product is mixed with pharmacologically inert, anorganic or organiccarriers, e.g. with lactose, sucrose, glucose, gelatine, malt, silicalgel, starch, or derivatives thereof, talkum, stearinic acid or itssalts, dried skim milk and the like.

For the production of soft capsules a carrier one may use for examplevegetable oils, petroleum, animal or synthetic oils, wax, fat, polyols.For the production of liquid solutions and syrups one may use carriersfor example water, alcohols, aqueous saline, aqueous dextrose, polyole,glycerin, vegetable oils, petroleum, animal or synthetic oils. For theproduction of suppositories one may use excipients as are e.g.vegetable, petroleum, animal or synthetic oils, wax, fat and polyols.For aerosol formulations one may use compressed gases suitable for thispurpose, as are e.g. oxygen, nitrogen, noble gas and carbon dioxide. Thepharmaceutically useful agents may also contain additives forconservation, stabilisation, e.g. UV stabilizer, emulsifier, sweetener,aromatiser, salts to change the osmotic pressure, buffers, coatingadditives and antioxidants.

Combinations with other therapeutic agents can include further agents,which are commonly used to treat cancer.

Compounds of Formula (IV), (V) and (VI) provided with suitableprotecting groups are produced as building blocks for the of compoundsof Formula (II). These can be linked via peptide coupling methods usingknown coupling reagents, e.g. hydroxybenzotriazole (HOBt) anddiisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC).

Building block (IV) can be assembled through peptide coupling ofcommercially available and known aminoacids.

Building block (V) can be assembled through a multicomponent reaction ofstarting materials of Formula (VII), (VIII) und (IX).

Herein PG is a known amino protecting group, for exampletert-butyloxycarbonyl (Boc). The resulting compound can be furthertransformed to building block (V) using R¹⁷COOCH₂Cl or H₂CO and R¹⁷COOHor H₂CO, TMS-Cl and R¹⁷COONa (I. Kornonen et al. Acta Chem. Scand. Ser.B 1982, 36(7), 467-474; R. Moriera et al. Tetrahedron Lett. 1994,35(38), 7107-7110; R. W. A. Luke, Tetrahedron Lett. 1996, 37(2),263-266).

Alternatively compounds of Formula (III) can be synthesized according tothe following scheme:

Building block (VI) of the following Formula:

can be steteoselectively synthesized using Evens reaction.

EXAMPLES

Synthesis of N-methyl-β-R,S-valine (1)

58.8 ml (0.47 mol) of a 8M methylamine solution in ethanol are slowlydropped to a solution of 33.8 g isobutyric aldehyde (0.47 mol) in 200 mlethanol while keeping the temperature in the flask below 5° C. Then 50ml THF are added and the mixture is refluxed for 1 h. Then 48.91 g (0.47mol) malonic acid is added in small portions and the mixture is refluxedfor 5 h. After cooling to 25° C. the precipitated is filtered off,washed with THF and dried under high vacuum. Yield: 50.34 gN-methyl-□-R,S-valine. Mass spectroscopy: expected molecular mass 145.2;found: m/z (M+H)⁺=146.1.

Synthese von N-Methyl-β-R,S-valinol (2)

14.5 g (0.1 mol) N-methyl-β-R,S-valine in 135 ml dry THF are addedslowly to 150 ml 1M lithiumaluminium hydrid in THF (0.15 mol) whilekeeping the temperature in the flask below 5° C. This mixture isrefluxed for 4 h. Subsequently the mixture is stirred over night. Themixture is hydrolized with 4 ml 12% KOH and 4 ml water. The precipitateis filtered off and is extracted two times with 80 ml hot THF. Thefiltrates are combined and the solvent is removed under vacuum. Theresulting oil is distilled (bp.: 48° C./0.5 mbar). Yield: 8.28 gN-methyl-β-R,S-valinol. Mass spectroscopy: expected molecular mass131.2; found: m/z (M+H)⁺=132.2.

Synthesis of N-methyl-β-R,S-valinolyl-tert.-butyldiphenyl-silylether (3)

2 g N-Methyl-β-R,S-valinole (15.24 mmol) are solubilized in 20 ml drydichlormethan together with 465.5 mg dimethylaminopyridin (3.81 mmol)and 2.66 ml triethylamine (19.05 mmol). To this solution 4.61 mltert.-butyldiphenylsilylchloride (18 mmol) is added and the mixture isstirred over night. 20 ml Water and 20 ml dichlormethane are added. Thewater phase is extracted two times with dichlormethane and the combinedorganic phases are dried over sodium sulfat. The sodium sulfate isfiltered of and the solvent is evaporated under vacuum. The residual oilis purified using column chromatography (eluent:ethylacetat/ethanol=8:2). Yield: 3.94 gN-nethyl-β-R,S-valinolyl-tert.butyldiphenylsilylether. Massspectroscopy: expected molecular mass 369.6; found: m/z (M+H)⁺=370.5.

Assembly of the dipeptide (R)-N-Boc-homoPro-(S,S)-Ile-OBzl (4)

7 g 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU) (21.81 mmol) and 2.4 ml N-methylmorpholin(21.81 mmol) are added to a solution of 5 g (R)-N-Boc-homoprolin (21.81mmol) in 40 ml dry DMF. After 10 minutes 7.21 g (S,S)-H-Ile-OBzl tosylat(18.32 mmol) and 2 ml N-methylmorpholin (18.32 mmol) are added. Thismixture is stirred over night at 25° C. and then 40 ml ethylacetate areadded. The organic layer is washed with saturated NaHCO₃. The aqueouslayer is extracted two times with ethylacetate. The combined organicextracts are washed with saturated NaCl and dried over Na₂SO₄. Thesolvent is evaporated under vacuum and the pure product appears. Yield5.54 g (R)-N-Boc-homoPro-(S,S)-Ile-OBzl. Mass spectroscopy: expectedmolecular mass 432.6; found: m/z (M+H)⁺=433.6

Boc-deprotection of (R)-N-Boc-homoPro-(S,S)-Ile-OBzl (5)

To a solution of (R)-N-Boc-HomoPro-(S,S)-Ile-OBzl in 60 ml dry THF isadded 120 ml 4M HCl in dioxan while keeping the temperature in the flaskbelow 5° C. After allowing the temperature to come to 20° C. the mixtureis stirred for 5 h. The solvent is evaporated and can be used directlywithout further purification for the next step. Yield: 4.1 g(R)-H-homoPro-(S,S)-Ile-OBzl. Mass spectroscopy: expected molecular mass332.5; found: m/z (M+H)⁺=333.6.

Reductive amination of (R)-H-homoPro-(S,S)-Ile-OBzl (6)

10 ml of a 37% formaldehyde solution (123 mmol) is added to 4.1 g(R)-homoPro-(S,S)-Ile-OBzl (12.3 mmol) in 20 ml methanol. The pH isadjusted to 5-6 with acetic acid and 1.932 g sodium cyanoborhydride(30.75 mmol) is added in portions. The mixture is stirred for 16 h at20° C. Subsequently the reaction is acidified with conc. HCl. Thesolvent is evaporated under vacuum and water is added. The pH isadjusted to pH 12 with solid NaOH and the mixture is extracted threetimes with dichlormethan. The organic layer is dried with Na₂SO₄ and thesolvent is evaporated. The resulting oil is evaporated by columnchromatography (eluent: ethylacetat:n-heptan=1:1). Yield: 3.9 g(R)-N-methyl-homoPro-(S,S)-Ile-OBzl. Mass spectroscopy: expectedmolecular mass 346.5; found: m/z (M+H)⁺=347.4

Hydration of (R)-N-methyl-homoPro-(S,S)-Ile-OBzl (7)

To a solution of 3.9 g (R)-N-methyl-homoPro-(S,S)-Ile-OBzl (11.26 mmol)in 30 ml methanol, 1.2 g Pd (10% C) are added. The flask is firstflushed with N₂ and then 10 min with H₂. Two more h the suspension isstirred under a H2-ballone; then the catalyst is filtered throughcelite, and washed two times with methanol. The solvent is evaporatedand the residual oil is lyophylized giving a white powder. Yield: 2.7 g(R)-N-methyl-homoPro-(S,S)-Ile-OH. Mass spectroscopy: expected molecularmass 256.4; found: m/z (M+H)⁺=257.4

Coupling of (R)-N-methyl-homoPro-(S,S)-Ile-OH andN-methyl-β-R,S-valinolyl-tert.butyldiphenylsilylether (8)

To a solution of 3.522 g (R)-N-methyl-homoPro-(S,S)-Ile-OH (13.74 mmol)in 15 ml dry DMF, 2.104 g hydoxybenzotriazol (13.74 mmol) and 2.151 mldiisopropylcarbodiimide (13.74 mmol) are added. After 15 minutesstirring 4.232 g N-methyl-β-R,S-valinolyl-tert.butyldiphenylsilylether(11.45 mmol) is added and the mixture is stirred for 16 h at 20° C. Theprecipitated diisopropyl urea is filtered off and the solvent isevaporated under vacuum. The residue is thoroughly stirred witdichlomethane and the residual diisopropyl urea is filtered off. Thedichlormethan solution is extracted with NaHCO3 and dryed subsequentlywith Na2SO4. After filtering off the Na2SO4 the solvent is evaporatedunder vacuum. The residue is purified with preparative HPLC. (RP-C18,eluent methanol+0.5% acetic acid/water+0.5% acetic acid). Yield: 3.91 g.Mass spectroscopy: expected molecular mass 608.0; found: m/z(M+H)⁺=609.0.

Deprotection of the tert.butyldiphenylsilyl Protecting Group of (8) (9)

3.91 g Of compound 8 (6.43 mmol) are solubilized in 30 ml drytetrahydrofuran and 2.223 ml tetrabutylammoniumfluorid (1M in THF) (7.72mmol) are added dropwise and the resulting mixture is stirred for 2 h at20° C. Then 8 ml of water is added and the tetrahydrofuran is evaporatedunder vacuum. The solution is neutralized and extracted five times withethylacetat. The combined organic phases are extracted two times withsaturated NaCl and dried over Na₂SO₄. The Na₂SO₄ is filtered off and thesolvent is evaporated. The resulting product is pure enough for furthertransformations. Mass spectroscopy: expected molecular mass 369.6;found: m/z (M+H)⁺=370.5.

Swern-Oxidation of (9) (10)

A solution of 0.665 ml oxalylchloride (7.75 mmol) in 25 ml drydichlormethan in a 250 ml flask is cooled to −70° C. under a N₂atmosphere. Slowly 1.188 ml dimethylsulfoxide (16.73 mmol) in 5 ml drydichlormethane is added in a way that the inner temperature is keptbelow −60° C. and the resulting mixture is stirred for 30 minutes at−70° C. Then a solution (6 ml) of (9) (6.43 mmol) in dichlormethane isadded in a way that the inner temperature is kept below −60° C. Afterstirring for further 30 minutes 4.459 ml triethylamin (32.17 mmol) areadded at −70° C. Once the flask reached 20° C., 15 ml water are addedand further 10 minutes are stirred. The aqueous phase is extracted twotimes with dichlormethan. The combined orgaic phases are dryed overNa₂SO₄, the Na₂SO₄ is filtered off and the solvent is evaporated. Theresulting product is pure enough to be used in the next step. Massspectroscopy: expected molecular mass 367.6; found: m/z (M+H)⁺=368.5.

Thiazolsynthesis (11)

0.695 ml Methylamin solution (33% in ethanol) (7.72 mmol) are added to(10) in 20 ml dry methanol and stirred for 1 h at 20° C. 991.3 mg3-Dimethylamino-2-isocyano-acrylacidmethylester (6.43 mmol) and 0.457 mlthioacetic acid (6.43 mmol) are added and stirred for 16 h at 20° C. Thesolvent is evaporate under vacuum and the residue is purified bypreparative HPLC (reversed phase-C18-phase, eluent methanol+0.5% aceticacid/water+0.5% acetic acid). Yield: 1.294 g. Mass spectroscopy:expected molecular mass 565.8; found: m/z (M+H)⁺=566.7

Saponification (11) (12)

To a solution of 1.294 g (11) (2.29 mmol) in 20 ml THF 220 mg LiOH (9.16mmol) in 20 ml water rare added and stirred for 16 h at 20° C. Thismixture is neutralized with 2N HCl. The solvent is evaporated underreduced pressure and the residue is purified with preparative HPLC(reversed phase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5%acetic acid). Yield: 1.14 g. Mass spectroscopy: expected molecular mass551.8; found: m/z (M+H)⁺=552.7

Coupling of (12) and □-aminodiphenylmethane (13)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 0.062 ml α-aminodiphenylmethan (0.36 mmol)are added. This mixture is stirred over night at 20° C., then thesolution is filtered and the solvent is evaporated under vacuum. Theresidue is purified by perperative HPLC (reversed phase-C18-phase,eluent methanol+0.5% acetic acid/water+0.5% acetic acid). Yield: 35 mg.Mass spectroscopy: expected molecular mass 717.0; found: m/z(M+H)⁺=718.1

Coupling of (12) and 3,3-diphenylpropylamine (14)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 76 mg 3,3-diphenylpropylamin (0.36 mmol)are added. This mixture is stirred over night at 20° C., then thesolution is filtered and the solvent is evaporated under vacuum. Theresidue is purified by perperative HPLC (reversed phase-C18-phase,eluent methanol+0.5% acetic acid/water+0.5% acetic acid). Yield: 35 mg.Mass spectroscopy: expected molecular mass 745.0; found: m/z(M+H)⁺=746.1.

Coupling of (12) and S-phenylalanine tert.butylester (15)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 24.3 mg S-phenylalanine tert.butylester(0.11 mmol) are added. This mixture is stirred over night at 20° C.,then the solution is filtered and the solvent is evaporated undervacuum. The residue is purified by perperative HPLC (reversedphase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5% aceticacid). Yield: 35 mg. Mass spectroscopy: expected molecular mass 755.0;found: m/z (M+H)⁺=756.2.

Coupling of (12) and S-tyrosin-O-tert.-butylether-tert.-butylester (16)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 32.3 mgS-tyrosin-O-tert.-butylether-tert.-butylester (0.11 mmol) are added.This mixture is stirred over night at 20° C., then the solution isfiltered and the solvent is evaporated under vacuum. The residue ispurified by perperative HPLC (reversed phase-C18-phase, eluentmethanol+0.5% acetic acid/water+0.5% acetic acid). Yield: 35 mg. Massspectroscopy: expected molecular mass 827.1; found: m/z (M+H)⁺=828.0.

Deprotection of (15) (17)

To a solution of 26 mg (15) (0.034 mmol) 2 ml dry dichlormethan 2 mltrifluoracetic acid are added. The mixture is stirred for 1 h and thesolvent is evaporated under the addition of n-heptan. The product ispure. Yield: 20 mg. Mass spectroscopy: expected molecular mass 698.9;found: m/z (M+H)⁺=699.5.

Deprotection of (16) (17)

To a solution of 26 mg (16) (0.034 mmol) 2 ml dry dichlormethan 2 mltrifluor acetic acid are added. The mixture is stirred for 1 h and thesolvent is evaporated under the addition of n-heptan. The product ispure. Yield: 18 mg. Mass spectroscopy: expected molecular mass 714.9;found: m/z (M+H)⁺=715.5.

Coupling of benzyloxycarbonyl-S-phenylalaninol and bromoaceticacid-tert.-butyl-ester (19)

To a solution of 1.141 g benzyloxycarbonyl-S-phenylalaninol (4 mmol) in20 ml dry THF 160 mg sodiumhydrid dispersion (60% in mineral oel) areadded. After end of H₂ evolution 1.182 ml bromo acetic acidtert.-butylester (8 mmol) are added and the mixture is stirred for 48 hat 20° C. The solvent is evaporated under reduced pressure and theproduct is purified with preparative HPLC (reversed phase-C18-phase,eluent methanol+0.5% acetic acid/water+0.5% acetci acid). Yield: 805 mg.Mass spectroscopy: expected molecular mass 399.5; found: m/z(M+H)⁺=400.3

Cbz-deprotection of (19) (20)

To a solution of 805 mg (19) (2.02 mmol) in 15 ml methanol, 800 mg Pd(10% C) are added. The flask is first flushed with N₂ and then stirred16 h under H2 atmosphere (2 H2 ballons). The catalyst is filteredthrough celite and washed several times with methanol. The solvent isevaporated. Yield: 482 mg. Mass spectroscopy: expected molecular mass265.4; found: m/z (M+H)⁺=266.3.

Coupling of (12) and (20) (21)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 16.8 mghydroxybenzotriazol hydrate (0.11 mmol) and 0.014 mldiisopropylcarbodiimid (0.11 mmol) are added. After stirring for 15minutes at 20° C. 29.2 mg (20) (0.11 mmol) are added. After stirringover night at 20° C. the solution is filtered and the residue ispurified by HPLC (reversed phase-C18-phase, eluent methanol+0.5% aceticacid/water+0.5% acetic acid). Yield: 22 mg. Mass spectroscopy: expectedmolecular mass 799.1; found: m/z (M+H)⁺=800.2.

Deprotection of (21) (22)

To a solution of 22 mg (21) (0.028 mmol) in 2 ml dry dichlormethan 2 mltrifluoracetic acid are added. This mixture is stirred for 1 h at 20° C.and the solvent is evaporated upon addition of n-heptan. The product ispure. Yield: 16 mg. Mass spectroscopy: expected molecular mass 757.0;found: m/z (M+H)⁺=758.2.

Coupling of (12) and methylamin (23)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 0.22 ml methylamin solution (2M in THF)(0.44 mmol) are added. This mixture is stirred over night at 20° C.,then the solution is filtered and the solvent is evaporated undervacuum. The residue is purified by perperative HPLC (reversedphase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5% aceticacid). Yield: 35 mg. Mass spectroscopy: expected molecular mass 564.8;found: m/z (M+H)⁺=565.7.

Coupling of (12) and R-Phenylalanintert.butylester (24)

To a solution of 49.5 mg (12) (0.09 mmol) in 3 ml dry DMF 18.6 mg6-chlorhydroxybenzotriazole (0.11 mmol) and 0.014 mldiisopropylcarbodiimide (0.11 mmol) are added. This mixture is stirredfor 15 minutes at 20° C. and 24.3 mg R-phenylalanine tert.butylester(0.11 mmol) are added. This mixture is stirred over night at 20° C.,then the solution is filtered and the solvent is evaporated undervacuum. The residue is purified by perperative HPLC (reversedphase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5% aceticacid). Yield: 35 mg. Mass spectroscopy: expected molecular mass 755.0;found: m/z (M+H)⁺=756.2.

Deprotection of (24) (25)

To a solution of 23 mg (24) (0.03 mmol) in 2 ml dry dichlormethan 2 mltrifluoracetic acid are added. The mixture is stirred for 1 h and thesolvent is evaporated under the addition of n-heptan. The product ispure. Yield: 18 mg. Mass spectroscopy: expected molecular mass 698.9;found: m/z (M+H)⁺=699.5.

Synthesis of N-formyl-S-valinol (26)

10 g S-Valinol (97 mmol) are dissolved in 50 ml ethylformiat andrefluxed for 1 h. The solvent is evaporated and the residue is destilledunder vacuum (bp.: 153° C./0.5 mbar). Yield: 8.4 g. Mass spectroscopy:expected molecular mass 131.2; found: m/z (M+H)⁺=132.3

Synthesis of N-methyl-S-valinol (27)

To a solution of 5.7 g lithiumaluminiumhydrid (150 mmol) in 200 ml dryTHF, 8.4 g N-formyl-S-valinol (64 mmol) dissolved in 40 ml dry THF areadded slowly and stirred for 16 h at 20° C. In several portions 30 gsodium sulfat decahydrat and 18 ml water are added and furthermorestirred for 3 h at 20° C. The solids are filtered off and the solvent isevaporated under vacuum. The residual material is purified bydestillation (bp.: 93° C./54 mbar). Yield: 3.7 g. Mass spectroscopy:expected molecular mass 1.17.2 found: m/z (M+H)⁺=118.1.

Synthesis of N-methyl-S-valinolyl-tert.butyldiphenylether (28)

To a solution of 1.64 g N-methyl-S-valinol (14 mmol) in 10 ml drydichlormethane 427 mg dimethylaminopyridine (3.5 mmol) and 2.44 mltriethylamin (17.5 mmol) are added. Then 4.3 ml tert.butyldiphenylsilylchloride are added and 16 h stirred at 20° C. Then 10 ml water and THFare added and the phases are separated. The aqueous phase id extractedtwo times with dichlormethane. The combined organic phases are driedover Na₂SO₄, subsequently the solvent is evaporated. The residue ispurified by column chromatography (eluent: ethylacetat/ethanol=8:2).Yield: 3.16 g. Mass spectroscopy: expected molecular mass 355.6 found:m/z (M+H)⁺=366.6

Coupling of (R)-N-methyl-homoPro-(S,S)-Ile-OH andN-methyl-S-valinolyl-tert.butyldiphenylsilylether (29)

To a solution of 1.54 g (R)-N-methyl-homoPro-(S,S)-Ile-OH (6 mmol) in 10ml dry DMF, 1.02 g 6-chlorohydroxybenzotriazol (6 mmol) and 0.939 mldiisopropylcarbodiimid (6 mmol) are added. The mixture is stirred for 15minutes and 2.56 g N-ethyl-S-valinolyl-tert.butyldiphenylether (7.2mmol) are added and stirred for 16 h at 20° C. Then the solvent isevaporated under vacuum and the residue is purified by preparative HPLC(reversed phase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5%acetic acid). Yield: 1.06 g. Mass spectroscopy: expected molecular mass593.9 found: m/z (M+H)⁺=594.8.

Cleavage of the tert.-butyldiphenylsilyl protecting group of (29) (30)

To a solution of 1.06 g (29) (1.79 mmol) in 10 ml dry THF a solution of2.15 ml tetrabutylammoniumfluorid (1M Lösung in THF) (2.15 mmol) isadded. The mixture is stirred for 16 h at 20° C. and then hydrolysedupon addition of 3 ml water. The organic solvent is evaporated and theaqueous phase is extracted five times with ethylacetat. The combinedorganic phases are washed with saturated Nail and dried over Na₂SO₄.After filtration of Na₂SO₄ the solvent is evaporated. Yield: 1.05 g(some residual silyl is remaining). Mass spectroscopy: expectedmolecular mass 355.5 found: m/z (M+H)⁺=356.5.

Swern-Oxidation of (30) (31)

0.316 ml Oxalylchlorid (1.98 mmol,) are solubilized in 3 ml drydichlormethan in a 100 ml flask under N₂ atmosphere and cooled to −70°C. To this solution 0.305 ml dimethylsulfoxid (4.29 mmol) in 0.6 mldichlormethan are added slowly (evolution of gas, keep the temperaturebelow −60° C.) and stirring continues for 30 minutes. A solution of 587mg (30) (1.65 mmol) in 2 ml dichlormethan is added while keeping thetemperature below −60° C. and stirring for 30 minutes. Then 1.146 mltriethylamin (8.25 mmol) is added. The mixture is allowed to come to 20°C. and then 10 ml water are added and the mixture is stirred for another10 minutes. The aqueous phase is extracted two times with dichlormethan.The combined organic layers are dried with Na₂SO₄. After filtering offthe Na₂SO₄ the solvent is evaporated. Yield: 636 mg. Mass spectroscopy:expected molecular mass 353.5 found: m/z (M+H)⁺=354.5.

Thiazolsynthesis (32)

636 Mg (31) (1.15 mmol) and 0.173 ml methylamin (33% in ethanol) (1.38mmol) in 3 ml dry methanol are stirred for 1 h at 20° C. 185 mg3-Dimethylamino-2-isocyano-acrylciacidmethylester (1.2 mmol) and 0.086ml thioacetic acid (1.2 mmol) are added and stirred for 16 h at 20° C.The solvent is evaporated and the residue is purified with preparativeHPLC (reversed phase-C18-phase, eluent methanol+0.5% aceticacid/water+0.5% acetic acid). Yield: 150 mg. Mass spectroscopy: expectedmolecular mass 551.8; found: m/z (M+H)⁺=552.7.

Saponification of (32) (33)

To a solution of 61 g (32) (0.11 mmol) in 2 ml THF, 10.6 mg LiOH (0.44mmol) in 2 ml water is added and stirred for 16 h at 20° C. The mixtureis neutralized with 2N HCl. The solvent is evaporated and the residue ispurified with preparative HPLC (reversed phase-C18-phase, eluentmethanol+0.5% acetic acid/water+0.5% acetic acid). Yield: 50 mg. Massspectroscopy: expected molecular mass 537.7; found: m/z (M+H)⁺=538.7.

Coupling of (33) and α-aminodiphenylmethane (34)

To a solution of 49.5 mg (33) (0.093 mmol) in 3 ml dry DMF, 14.2 mghydroxybenzotriazol (0.093 mmol) and 0.012 ml diisopropylcarbodiimid(0.093 mmol) are added and stirred for 15 minutes at 20° C. 0.064 mlα-aminodiphenylmethan (0.372 mmol) is added and is stirred over night.The mixture is filtered and evaporated and the residue is purified bypreparative HPLC (reversed phase-C18-phase, eluent methanol+0.5% aceticacid/water+0.5% acetic acid). Yield: 30 mg. Mass spectroscopy: expectedmolecular mass 703.0; found: m/z (M+H)⁺=704.1.

General Procedure for the Synthesis of Thiazoles

1 Mmol of the carbonyl compound (IX) is solubilized in 3 ml dry THFgelost under N₂ atmosphere and 1 mmol borontrifluorid etherat are added.After 10 min 1 mmol of isocyanide (VIII) and 1 mmol of thioacarboxylicacid (VII) are added and stirred for 72 h. Water is added and optinallyfiltered through celite. The solvent is evaporated under vacuum. Theresidue is solubilized in ethylacetate. The organic phase is washed twotimes with water. After drying the organic phase over Na₂SO₄ the solventis evaporated. The residue is purified by preparative HPLC (reversedphase-C18-phase, eluent methanol+0.5% acetic acid/water+0.5% aceticacid).

Compounds of Formula (IX) can be synthesized for example by aα-aminoalkylation of isobutyric aldehyd, ammoniumacetat or a primaryamine or amine hydrochlorid and malonic acid:

The resulting β-amino acid can be subsequently N-alkylated (e.g. byreductive amination) and protected (e.g. t-butyloxycarbonyl, Boc). Thenthe carboxylic acid group is transformed to the aldehyde (e.g. byreduction to the alkohol by LiAlH₄ and subsequent Swern oxidation to thealdehyde; see for example R. C. Larock, Comprehensive OrganicTransformations, VCH Publishers, New York, 1989). Alternatively theβ-aminoacid can be synthesized by a Arndt-Eistert procedure startingfrom valine.

Example 35

C₁₉H₃₀N₂O₆S (414.5248)

MS (ESI): 415 [M+H]

Example 36

C₂₄H₃₂N₂O₆S (476.5964)

MS (ESI): 477 [M+H]

Example 37

C₂₈H₄₇N₃O₈S (585.7661)

MS (ESI): 586 [M+H]

Example 38

A compound from example 35 (0.1 mmol) is stirred in 2 ml dichlormethan(DCM) and 0.1 ml trifluoracetic acid (TFA) for 1 h at 20° C. Theliquides DCM/TFA are evaporated and the residue is purified by HPLC.

C₁₄H₂₂N₂O₄S (314.4064)

MS (ESI): 315 [M+H]

Example 39

The compound from example 37 (0.1 mmol) is dissolved in 2 mlDichloromethan (DCM) and 0.1 ml trifluoracetic acid (TFA) is added andstirred for 1 h at 20° C. The liquides DCM/TFA are evaporated and theresidue is purified by HPLC.

C₁₈H₃₁N₃O₄S (385.5295)

MS (ESI): 386 [M+H]

Bispiel 40:

C₁₈H₂₈N₂O₆S (400.4977)

MS (ESI): 401 [M+H]

Example 41

1 mmol of the compound from example 40 in 1 ml methanol is stirred with1 ml 4 M ammonia solution in methanol for 2 h at 20° C. Tsolvent isevaporated under vacuum.

C₁₆H₂₆N₂O₅S (358.4600) MS (ESI): 381 [M+Na]

Examples 42 and 43 Ester Coupling of Hydroxythiazols (Example 41) andDipeptide (7) and Subsequent Transacylation

To 2 Mmol (512 mg)3-methyl-2-[(1-methyl-piperidin-2-carbonyl)-amino]-pentanoic acid (7) in5 ml dry dichlormethan is added 2 mmol (252 mg)N,N′-diisopropylcarbodiimide (DIC) in 2.5 ml DCM and 0,2 mmol (24 mg)DMAP in 2.5 ml DCM under N₂ atmosphere at 0° C. The mixture is stirred 5minutes at 0° C. 1 mmol (372 mg)2-[3-(tert.-butoxycarbonyl-methylamino)-1-hydroxy-4-methyl-pentyl]-thiazole-4-carboxylicacid methylester (example 41) is dissolved in 5 ml DCM and slowly addedvia syringe. The mixture is stirred 4 h at 20° C. The mixtureconcentrated in vacuum and the precipitated urea is filtered off. To thefiltrate is added 1 ml of trifluoracetic acid and 1 h stirred at 20° C.and the solvents are evaporated under vacuum. The residue is dissolvedin 1 ml dry dichlormethan and 1 ml triethylamin is added and 1 h stirredat 20° C. The solvent is evaporated under vacuum. The rearrangedcoupling product is purified by HPLC.

2-(3-(tert.-butoxycarbonyl-methyl-amino)-4-methyl-1-{3-methyl-2-[(1-methyl-piperidin-2-carbonyl)-amino]-pentanoyloxy}-pentyl)-thiazol-4-carboxylicacid methylester (42)

C₃₀H₅₀N₄O₇S (610,82)

MS (ESI): 611 [M+H]; 633 [M+Na]

2-[1-Hydroxy-4-methyl-3-(methyl-{3-methyl-2-[(1-methyl-piperidin-2-carbonyl)-amino]-pentanoyl}-amino)-pentyl]-thiazol-4-carboxylicacid methylester (43)

C₂₅H₄₂N₄O₅S (510,70)

MS (ESI): 511 [M+H]; 533 [M+Na]

Examples 44 and 45 Reaction of (43) and Phenylethylamine and SubsequentAcetylation

0.14 Mmol (72 mg)2-[1-hydroxy-4-methyl-3-(methyl-{3-methyl-2-[(1-methylpiperidine-2-carbonyl)-amino]-pentanoyl}-amino)-pentyl]-thiazol-4-carboxylicacid methylester (43) are stirred with 100 μl phenylethylamine for 12 hat 20° C. The reaction mixture is filtered through a plug of silica geland washed with ethylacetate. The mixture is evaporated to dryness and40 μl acetic acid anhydride and 10 μl pyridine are added. The mixture isstirred for 2 h at 20° C. A third of the reaction mixture is purifiedwith a analytical HPLC.

1-methyl-piperidin-2-carbonsäure-[1-({1-[2-hydroxy-2-(4-phenethylcarbamoyl-thiazol-2-yl)-ethyl]-2-methyl-propyl}-methylcarbamoyl)-2-methyl-butyl]-amide(44)

C₃₂H₅₄₉N₅O₄S (599,84)

MS (ESI): 600 [M+H]; 622 [M+Na]

Acetic acid4-methyl-3-(methyl-{3-methyl-2-[(1-methyl-piperidine-2-carbonyl)-amino]-pentanoyl}-amino)-1-(4-phenethylcarbamoyl-thiazol-2-yl)-pentylester (45)

C₃₄H₅₁N₅O₅ S (641,88)

MS (ESI): 642 [M+H]; 664 [M+Na]

Synthesis of Building Block (VI) According to Evans-Procedure

(2S)-2-Phthalimido-3-phenyl-propanol

To L-phenylalaninol (1.0 g, 6.61 mmol) and Na₂CO₃ (1.05 g, 9.92 mmol) ina 1:1 mixture of THF (10 mL) and H₂O (10 mL) N-carbethoxyphthalimide(1.74 g, 7.94 mmol) is added and stirred 4 h at 20° C. To this reactionmixture ethylacetate (20 mL) is added. The aqueous phase is extractedtwo times with 15 mL ethylacetate and the combined organic phases arewashed with saturated NaCl, dried with Na₂SO₄ and the solvent isevaporated under vacuum. The product is purified with columnchromatography using 2% MeOH in CH₂Cl₂. Yield: 1.41 g (76%); MS (ESI)282 [M+H]; ¹H NMR (300 MHz, CDCl₃): δ 7.82-7.76 (m, 2H), 7.73-7.66 (m,2H), 7.24-7.12 (m, 5H), 4.70-4.58 (m, 1H), 4.12-4.02 (m, 1H), 3.98-3.88(m, 1H), 3.20 (d, J=12.5 Hz, 2H), 2.80-2.72 (m, 1H).

(2S)-1-Trifluoromethanesulfonyl-2-phthalimido-3-phenyl propanoat

To a solution of (2S)-2-phthalimido-3-phenylpropanole (0.42 g, 1.49mmol) in dry CH₂Cl₂ (5 mL), pyridin (146 μL, 1.79 mmol) is added at −78°C. and stirred for 20 minutes. To this mixture 3 mintrifluoromethansulfonic acid anhydride (264 μL, 1.57 mmol) is added inbetween 3 minutes and stirred for 1 h at −78° C. The reaction mixture isquenched with 3 ml saturated NaCl. The aqueous phase is extracted with 5mL of CH₂Cl₂, the combined organic phases are washed with 5 ml saturatedNaCl gewaschen, dried with Na₂SO₄ and the solvent is evaporated. Theproduct is purified with column chromatography using 20% ethylacetat inhexen. Yield: 0.41 g (66%). MS (ESI) 414 [M+H]; ¹H NMR (300 MHz, CDCl₃):δ 7.84-7.77 (m, 2H), 7.75-7.68 (m, 2H), 7.28-7.14 (m, 5H), 5.18 (t,J=13.0 Hz, 1H), 5.00-4.85 (m, 1H), 4.55-4.30 (m, 1H), 3.40-3.25 (m, 2H).

Evans Alkylation

(4R)-3-propanoyl-4-benzyl-2-oxazolidinone (0.100 g, 0.43 mmol) isdissolved in 2 ml dry THF in an argon atmosphere and subsequently cooledto −40° C. LiHMDS (1M/THF) (0.47 mL, 0.47 mmol) is added and stirred for45 minutes.(2S)-1-Trifluoromethansulfonyl-2-phthalimido-3-phenylpropanoate (0.266g, 0.64 mmol) in dry THF (2 mL) is added. The mixture is stirred for 4 hat −40° C. and subsequently quenched with 3 ml saturated NaCl. Theaqueous phase is extracted 2 times with 5 ml ethylacetate. The combinedorganic phases are washed with 3 ml saturated NaCl, dried with Na₂SO₄and the solvent is evaporated under vacuum. The product is purified withcolumn chromatography using 25% ethylacetate in hexen. Yield: 0.149 g(70%). The diastereomers can be separated using preparative TLC. Thewanted diastereomer is formed in excess: 8:2.

(2′S,4′R,4R,)-3-(2′Methyl-4′phthalimido-5′phenylpentanoyl)-4-benzyl-1,3-oxazolidin-2-one (Major Product)

MS (ESI): 497 [M+H]; ¹H NMR (300 MHz, CDCl₃): δ 7.77 (t, J=8.5 Hz, 2H),7.63 (t, J=8.4 Hz, 1H), 7.55 (t, J=8.4 Hz, 1H), 7.42 (d, J=8.5 Hz, 2H),7.37-7.22 (m, 6H), 7.10 (d, J=8.6 Hz, 2H), 5.08 (q, J=9.6 and 16.1 Hz,1H), 4.56-4.42 (m, 2H), 4.20-4.00 (m, 4H), 3.45 (dd, J=10.7 and 16.1 Hz,1H), 3.12-2.98 (m, 2H), 2.34 (dd, J=12.8 and 13.9 Hz, 1H), 1.62 (d,J=8.6 Hz, 3H).

(2′R,4′R,4R,)-3-(2′Methyl-4′phthalimido-5′phenylpentanoyl)-4-benzyl-1,3-oxazolidin-2-one (Minor Product)

MS (ESI): 497 [M+H]; ¹H NMR (300 MHz, CDCl₃): δ 8.12 (d, J=8.6 Hz, 1H),7.76 (d, J=8.5 Hz, 1H), 7.63 (t, J=8.6 Hz, 1H), 7.53 (t, J=8.5 Hz, 1H),7.40-7.20 (m, 10H), 5.10 (q, J=7.5 and 15.0 Hz, 1H), 4.94-4.84 (m, 1H),4.54-4.42 (m, 1H), 4.36-4.08 (m, 4H), 3.46-3.30 (m, 2H), 3.12 (dd, J=9.6and 11.8 Hz, 1H), 2.88 (dd, J=9.5 and 12.8 Hz, 1H), 1.00 (d, J=9.6 Hz,3H).

Cleavage of the oxazolidinons: Evans et. al., J. Am. Chem. Soc. 1982,104, 1737-1739.

Deprotection of the phthalimids: using hydrazine/EtOH at 20° C.: Sasaki,T. et. al., J. Org. Chem. 1978, 43, 2320; Khan, M. N. et. al., J. Org.Chem. 1995, 60, 4536.

According to the herein disclosed synthetic procedures also thefollowing tubulysin derivatives where synthesized:

The following residues where used:

-   m=0, 1, 2, 3;-   R¹=methyl, ethyl;-   R⁶=isopropyl, isobutyl, ethyl, cyclopropyl, CH₂-cyclopropyl,    CH(CH₃)CH₂CH₃;-   R⁹=isopropyl, trifluormethyl, chlormethyl, isobutyl, ethyl,    cyclopropyl, CH₂-cyclopropyl, CH(CH₃)CH₂CH₃, cyclopentyl,    cyclohexyl;-   R¹⁷=methyl, ethyl, propyl, isopropyl, butyl, isobutyl, CH═C(CH₃),    cyclopropyl, cyclobutyl, cyclohexyl;-   R²⁰=methyl, ethyl, propyl, isopropyl, phenyl;-   R¹⁹=

1. A compound of the following general formula:

wherein: A represents an optionally substituted 5- or 6-memberedheteroaryl ring X is O, S or a group of Formula NR¹³ or CR¹⁴R¹⁵; Y is O,S or a group of Formula NR¹⁶ and R¹, R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹³,R¹⁴, R¹⁵ and R¹⁶ are independently of each other H, alkyl, alkenyl,alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl,heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl, R²and R³ together constitute a group of Formula (CH₂)n wherein n is 4; andR⁸ is hydrogen or alkyl; R¹² is H, alkyl, alkenyl, alkynyl, heteroalkyl,aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl,heterocycloalkyl, aralkyl, heteroaralkyl or NR¹⁸R¹⁹; R¹⁸ is H or methyl;and R¹⁹ is aralkyl or heteroaralkyl or a pharmacologically acceptablesalt, solvate, hydrate or a pharmacologically acceptable formulationthereof.
 2. A compound of claim 1, wherein A has the followingstructure:


3. The compound according to claim 1 wherein R¹² is NR¹⁸ R¹⁹; R¹⁸ is Hor methyl; R¹⁹ has the formula:

wherein: R²¹ is —OH, —NH₂, alkyloxy, alkylamino or dialkylamino; R²² ishalogen, —OH, —NO₂, —NH₂, alkyloxy, alkylamino or dialkylamino; and p is0, 1, 2 or
 3. 4. A compound of claim 1 wherein X is a CH₂ group.
 5. Acompound of claim 1 wherein Y is O.
 6. A compound of claim 1 wherein R¹is C₁-C₄ alkyl.
 7. A compound of claim 1 wherein R⁴ is H or methyl.
 8. Acompound of claim 1 wherein R⁵ is H.
 9. A compound of claim 1 wherein R⁶is C₁-C₆ alkyl, C₃-C₆ cycloalkyl or C₄-C₇ alkylcycloalkyl.
 10. Acompound of claim 1 wherein R⁷ is H or methyl.
 11. A compound of claim 1wherein R⁹ is C₁-C₆ alkyl.
 12. A compound of claim 1 wherein R¹⁰ is H ormethyl.
 13. A compound of claim 1 wherein R¹¹ is H or a group of Formula(C═O)—(C₁₋₄)alkyl.
 14. A compound of claim 1 wherein R¹² is a group ofFormula NR¹⁸R¹⁹, wherein R¹⁸ is H or methyl and R¹⁹ is aralkyl orheteroaralkyl.
 15. A pharmaceutical composition comprising a compound ofclaim 1 and optionally one or more carriers and/or adjuvants.
 16. Amethod for treating a patient suffering from colon adenocarcinoma,breast adenocarcinoma, ovarian adenocarcinoma, epidermoid adenocarcinomaor prostate adenocarcinoma, comprising administering to the patient oneor more compounds of claim
 1. 17. The method of claim 16 wherein thepatient is identified as suffering from colon adenocarcinoma, breastadenocarcinoma, ovarian adenocarcinoma, epidermoid adenocarcinoma orprostate adenocarcinoma, and the one or more compounds are administeredto the identified patient.